Caffeine, a cardiac stimulant and mild diuretic, is one of the world's most widely used drugs. Caffeine originates from the chemical family methylxanthine or xanthine which occur naturally in some plants. Beverages which are made from the nuts, leaves, stems, bark, of such caffeine containing plants are major sources of natural caffeine such as coffee made from the Coffea arabica plant and other caffeine containing plants, soft drinks including diet drinks made from Kola nuts, tea made from the leaves of Thea sinensis and cocoa used to make chocolate.
Caffeine is a food additive regulated by the U.S. Federal Food and Drug Administration (“FDA”) and as such manufacturers of food and beverages are required to place the name caffeine on the food product label when caffeine has been added to the food product to prepare a caffeinated product. The FDA also regulates caffeine's use as a stimulant in some over the counter and prescription medicines.
The caffeine content of coffee, tea, soft drinks and medications containing caffeine varies widely. For example, an average five ounce cup of percolated coffee contains from about 40 mg to about 170 mg caffeine (Spiller, G. A., Caffeine (1998) CRC Press, Table 9, p. 214).
Whether due to increased focus on health or to the potential pharmacological effect of the consumption of caffeine, many people desire to know, at the time or prior to consumption of a fluid suspected of containing caffeine, whether or not that consumable fluid contains caffeine.
Currently, caffeine is measured by a variety of methods including ultraviolet spectroscopy, thin-layer chromatography, gas chromatography, high-performance liquid chromatography and capillary electrophoresis (Spiller, G. A. (Ed.), Caffeine (1998) 13-33, CRC Press, New York. However, none of these methods are readily applicable to home or restaurant use.
There are caffeine specific antibodies available, both rabbit polyclonal and mouse monoclonal. These traditional antibodies comprise heavy and light chains and have been shown to irreversibly denature at high temperatures (Van Der Linden, R. H. J., et al., Biochim, Biophys. Acta (1999); 1431:37-46; Ewert, S., et al., Biochemistry (2002); 41:3628-3636). These antibodies would not be applicable for caffeine determination at high temperature which may be convenient in some analytical settings.
Members of the Camelidae family have been shown to produce a form of antibody that is devoid of light chains (Hamers-Casterman, C., et al., Nature (1993) 363:446-448). The variable domains of such heavy-chain only antibodies (VHH) have been cloned from peripheral blood lymphocytes from camel (Camelus dromedarius) (Ghahroudi, M. A., et al., FEBS Lett. (1997) 414:521-526) and llama (lama glama) (Van Der Linden, R., et al., J Immunol Meth (2000); 240:185-195). These single domain antibody fragments can refold (Pérez, J. M. J., et al., Biochemistry (2001) 40:74-83) and maintain functionality after thermal dissociation (van der Linden, R. H. J., et al., Biochimica et Biophysica Acta (1999) 1431:37-46; Ewert, S., et al., Biochemistry (2002) 41:3628-3636). In some cases, VHH fragments can bind specifically at temperatures up to 90° C. (Van Der Linden, R. H. J., supra (1999)). In addition, there are reports of hapten specific VHH fragments (Frenken. L. G. J., et al., J Biotech (2000); 78:11-21; Spinelli, S., et al., Biochemistry (2000) 39:1217-1222).
It is often convenient to test for caffeine at a high temperature and in this case reagents must be stable over a range of temperatures and provide accurate caffeine determination even if the detector system is heated.
Despite research in this area to accomplish these objectives, there remains a need for a dynamic, on location, “kit type,” easy to use, straightforward, visual system to measure caffeine content of fluids, including hot fluids.